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Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line.

Yue Z, Carvalho A, Xu Z, Yuan X, Cardinale S, Ribeiro S, Lai F, Ogawa H, Gudmundsdottir E, Gassmann R, Morrison CG, Ruchaud S, Earnshaw WC - J. Cell Biol. (2008)

Bottom Line: However, these cells show normal sensitivity to the chemotherapeutic agent etoposide.Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth.As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, Scotland, UK.

ABSTRACT
Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

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The BIR domain is crucial for Survivin function. (A) Location of C86 in the CPC form of Survivin. (B) Expression of SurvivinC86A-GFP and loss of wild-type (WT) Survivin expression from cultures grown in doxycycline. (C) SurvivinC86A-GFP fails to rescue the life of SurvivinOFF cells; cells expressing this protein as their sole form of Survivin die with kinetics essentially identical to those of SurvivinOFF cells. (D) SurvivinC86A-GFP fails to localize in SurvivinON/OFF cells. INCENP localization is normal in SurvivinON cells, so SurvivinC86A-GFP fails to act as a dominant-negative mutant. Panel a shows the normal localization of Survivin-GFP in a control experiment. (E) Location of C59 in the Survivin dimer. (F) Expression of SurvivinC59A-GFP and loss of wild-type Survivin expression from cultures grown in doxycycline. (G) Survivin C59A-GFP fails to rescue the life of SurvivinOFF cells. (H) SurvivinC59A-GFP fails to localize in SurvivinON/OFF cells. INCENP localization is normal in SurvivinON cells, so SurvivinC59A-GFP also fails to act as a dominant-negative mutant. Bars, 5 μm.
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fig6: The BIR domain is crucial for Survivin function. (A) Location of C86 in the CPC form of Survivin. (B) Expression of SurvivinC86A-GFP and loss of wild-type (WT) Survivin expression from cultures grown in doxycycline. (C) SurvivinC86A-GFP fails to rescue the life of SurvivinOFF cells; cells expressing this protein as their sole form of Survivin die with kinetics essentially identical to those of SurvivinOFF cells. (D) SurvivinC86A-GFP fails to localize in SurvivinON/OFF cells. INCENP localization is normal in SurvivinON cells, so SurvivinC86A-GFP fails to act as a dominant-negative mutant. Panel a shows the normal localization of Survivin-GFP in a control experiment. (E) Location of C59 in the Survivin dimer. (F) Expression of SurvivinC59A-GFP and loss of wild-type Survivin expression from cultures grown in doxycycline. (G) Survivin C59A-GFP fails to rescue the life of SurvivinOFF cells. (H) SurvivinC59A-GFP fails to localize in SurvivinON/OFF cells. INCENP localization is normal in SurvivinON cells, so SurvivinC59A-GFP also fails to act as a dominant-negative mutant. Bars, 5 μm.

Mentions: In human Survivin, four conserved residues, Cys57, Cys60, His77, and Cys84, form a zinc finger that stabilizes the structure of the BIR domain (Chantalat et al., 2000; Verdecia et al., 2000). To confirm the role of the BIR domain in Survivin function in vivo, SurvivinC86A (equivalent to hSurvivinC84A) and SurvivinC59A (equivalent to hSurvivinC57A) mutants were stably transfected into KO1, where they were expressed at levels comparable with the rescue Survivin (Fig. 6, B and F).


Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line.

Yue Z, Carvalho A, Xu Z, Yuan X, Cardinale S, Ribeiro S, Lai F, Ogawa H, Gudmundsdottir E, Gassmann R, Morrison CG, Ruchaud S, Earnshaw WC - J. Cell Biol. (2008)

The BIR domain is crucial for Survivin function. (A) Location of C86 in the CPC form of Survivin. (B) Expression of SurvivinC86A-GFP and loss of wild-type (WT) Survivin expression from cultures grown in doxycycline. (C) SurvivinC86A-GFP fails to rescue the life of SurvivinOFF cells; cells expressing this protein as their sole form of Survivin die with kinetics essentially identical to those of SurvivinOFF cells. (D) SurvivinC86A-GFP fails to localize in SurvivinON/OFF cells. INCENP localization is normal in SurvivinON cells, so SurvivinC86A-GFP fails to act as a dominant-negative mutant. Panel a shows the normal localization of Survivin-GFP in a control experiment. (E) Location of C59 in the Survivin dimer. (F) Expression of SurvivinC59A-GFP and loss of wild-type Survivin expression from cultures grown in doxycycline. (G) Survivin C59A-GFP fails to rescue the life of SurvivinOFF cells. (H) SurvivinC59A-GFP fails to localize in SurvivinON/OFF cells. INCENP localization is normal in SurvivinON cells, so SurvivinC59A-GFP also fails to act as a dominant-negative mutant. Bars, 5 μm.
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Related In: Results  -  Collection

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Show All Figures
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fig6: The BIR domain is crucial for Survivin function. (A) Location of C86 in the CPC form of Survivin. (B) Expression of SurvivinC86A-GFP and loss of wild-type (WT) Survivin expression from cultures grown in doxycycline. (C) SurvivinC86A-GFP fails to rescue the life of SurvivinOFF cells; cells expressing this protein as their sole form of Survivin die with kinetics essentially identical to those of SurvivinOFF cells. (D) SurvivinC86A-GFP fails to localize in SurvivinON/OFF cells. INCENP localization is normal in SurvivinON cells, so SurvivinC86A-GFP fails to act as a dominant-negative mutant. Panel a shows the normal localization of Survivin-GFP in a control experiment. (E) Location of C59 in the Survivin dimer. (F) Expression of SurvivinC59A-GFP and loss of wild-type Survivin expression from cultures grown in doxycycline. (G) Survivin C59A-GFP fails to rescue the life of SurvivinOFF cells. (H) SurvivinC59A-GFP fails to localize in SurvivinON/OFF cells. INCENP localization is normal in SurvivinON cells, so SurvivinC59A-GFP also fails to act as a dominant-negative mutant. Bars, 5 μm.
Mentions: In human Survivin, four conserved residues, Cys57, Cys60, His77, and Cys84, form a zinc finger that stabilizes the structure of the BIR domain (Chantalat et al., 2000; Verdecia et al., 2000). To confirm the role of the BIR domain in Survivin function in vivo, SurvivinC86A (equivalent to hSurvivinC84A) and SurvivinC59A (equivalent to hSurvivinC57A) mutants were stably transfected into KO1, where they were expressed at levels comparable with the rescue Survivin (Fig. 6, B and F).

Bottom Line: However, these cells show normal sensitivity to the chemotherapeutic agent etoposide.Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth.As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, Scotland, UK.

ABSTRACT
Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

Show MeSH
Related in: MedlinePlus